In recent years, gene expression analysis using DNA chips (also referred to as DNA microarrays hereinafter) is done in various fields including drug development. Different specimen DNAs are made to react with a DNA microarray in which various kinds of gene sets (probes) are arranged. Gene dosages which exist in the respective specimens are compared. Genes which are present in high dosages (the expression amounts are large) or inactive genes (the expression amounts are small) at each stage are classified and analyzed in association with functions.
An example is an infectious etiologic agent test. In Japanese Patent Laid-Open No. 2001-299396, Ezaki et al have proposed a microorganism identification method using, as a DNA probe, a DNA chip on which chromosome DNAs are immobilized. According to this method, a plurality of chromosome DNAs originated in a plurality of known microorganisms with different GC contents are made to react with chromosome DNAs originated in an unknown microorganism in a specimen. When the resultant hybridization complex is detected, the unknown microorganism in the specimen can be detected.
As probes used for DNA chips for infectious etiologic agent tests, Ono et al have proposed a bacterial detection probe using restriction enzyme fragments in Japanese Patent Laid-Open No. 6-133798, a Pseudomonas aeruginosa detection probe in Japanese Patent Laid-Open No. 10-304896, and a detection probe using restriction enzyme fragments of Escherichia coli, klebsiella pneumoniae, and Enterobacter cloacae in Japanese Patent Laid-Open No. 10-304897.
As a microarray, for example, a microarray using stamping called a Stanford method is known. For example, DNA chips on which cDNA fragments of known genes of human origin, which are related to cancers, are applied by spotting or stamping and chips prepared by bonding cDNA fragments of 1,000 kinds of known genes of human origin to slide glasses are commercially available from TAKARA SHUZO.
On the other hand, a chip available from Affymetrix is prepared by designing an oligonucleotide probe set on the basis of the known gene cDNAs, and probes are laid out by synthesis on a substrate. Oligoprobes are laid out on one chip at a high density so that the expression levels of 10,000 or more genes can be analyzed at once.
However, the DNA chips of the prior arts described above use DNA probes such as chromosome DNAs or restriction enzyme fragments. DNAs directly extracted from microorganisms are used as materials. For this reason, the chips can hardly be mass-prepared at a time and are not suitable for clinical diagnosis. For application to clinical diagnosis, mass production of inexpensive and uniform DNA chips is necessary. For this purpose, mass preparation of uniform DNAs as probe solutions is essential. However, mass preparation of DNA probes is impossible. Even for DNA probes, when PCR amplification reaction is used, the number of DNAs can gradually be increased. However, mass preparation at a time using the PCR reaction is difficult, and the DNA chips are difficult to use for clinical diagnosis.
In addition, since the base length of a DNA probe is large, it is difficult to identify one species in similar species. Such a DNA probe is therefore not suitable for, e.g., infection detection. In treating an infection, the species must be specified, and antibiotic drugs corresponding to it must be selected and administered. For this purpose, an infection detection probe is required to have a function capable of detecting a species while discriminating similar species, although bacteria belonging to the same species need not accurately be discriminated (that is, bacteria in the same species can be detected all together). However, in, e.g., the DNA chip using restriction enzyme fragments of Escherichia coli, klebsiella pneumoniae, and Enterobacter cloacae, which is disclosed in Japanese Patent Laid-Open No. 10-304897, cross reaction occurs between the three species because of the large base length of the probe. Since similar species cannot individually be discriminated, the DNA chip can hardly be used for infection detection.
As an application purpose of microarrays, infectious etiologic agent tests have received a great deal of attention. Some probe sets aiming at testing infectious etiologic agents have also been proposed.
As an important point of bacterial tests using microarrays, detection must be possible even when the number of infectious etiologic agents is small. To do this, it is effective to amplify specific parts in the base sequences of the DNAs of infectious etiologic agents by, e.g., PCR reaction using primers. For example, a 16s rRNA gene arrangement contains a sequence unique to the species in the information of about 1,700 base pairs. When the sequence is used, classification can be done to some extent. In detecting/identifying bacteria, 16s rRNA parts in the DNA base sequences of bacteria are preferably used. Hence, it is demanded to amplify the 16s rRNA parts.
For various kinds of bacteria, however, the gene arrangements are only partially clarified, and the 16s rRNAs are not totally known. For this reason, it is not easy to design primers for PCR amplification reaction.